The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, ...frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid.
Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying bla
and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems.
We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.
Abstract
Background
Carbapenem-resistant Enterobacterales (CRE) are a major public health threat, largely due to the presence of carbapenemases, which are globally disseminated in mobile genetic ...elements. The emergence of CRE carrying multiple carbapenemases has been reported in several countries, particularly after the COVID-19 pandemic. In this study, we report the emergence of dual-producing CRE (DP-CRE) in Chile and provide a phenotipic and genomic characterization using whole-genome sequencing (WGS).
Methods
We evaluated the presence of carbapenemase in a total of 1367 CRE isolates recovered from invasive infections in 11 healthcare centers since 2018. Among them, 9 DP-CRE were detected and included in this report. Antimicrobial susceptibility was tested by broth microdilution and disk diffusion methods (CLSI, 2023), while blaKPC, blaVIM, blaIMP and blaNDM genes were detected by PCR. WGS was carried out using short and long reads (Illumina and Oxford Nanopore) and hybrid assemblies were performed.
Results
All 9 DP-CRE identified were recovered between November 2021 and June 2022 from 3 healthcare centers of a single city. In terms of species, 6 were identified as E. coli, one isolate of K. pneumoniae, one K. oxytoca and one C. freundii. All DP-CRE identified carried the combination of blaKPC and blaNDM. Genomic analyses confirmed all but one isolate carried blaKPC-2 and blaNDM-7. The remaining genome belonged to a K. pneumoniae that harboured blaKPC-3 and blaNDM-7. All 9 isolates exhibited resistance to all β-lactams, including carbapenems, aztreonam (ATM), cephalosporins and β-lactam/β-lactamase inhibators. Cefiderocol (FDC) was the only compound active against all the isolates. Also, all the DP-CRE became susceptible to ATM when combined with ceftazidime/avibactam (CZA). Hybrid assemblies revealed that blaKPC and blaNDM were harboured on independent plasmids (∼58,900 bp and ∼41,100 bp, respectively) as shown in Figure 1.
Conclusion
To the best of our knowledge, this is the first report of the emergence of DP-CRE in Chile after COVID-19 pandemic. Our results highlight the relevance of active surveillance of multidrug-resistance pathogens. FDC and CZA/ATM were the only compounds that remained active in vitro against these pathogens.
Disclosures
All Authors: No reported disclosures
Resumen Introducción: La aparición y diseminación de Enterobacterales resistentes a carbapenémicos ha generado un gran impacto en las infecciones asociadas a la atención de salud en el mundo. ...Recientemente, en Chile se detectó un brote por Klebsiella pneumoniae productora de carbapenemasas tipo oxacilinasas (OXA) de la subfamilia tipo OXA-48, reportándose los primeros casos en pacientes hospitalizados mayoritariamente en la zona norte del país. Objetivo: Determinar los perfiles fenotípicos, genotípicos y de susceptibilidad antimicrobiana de 16 cepas referidas durante mayo del año 2021 desde las regiones de Antofagasta y Metropolitana al Laboratorio de Referencia del Instituto de Salud Pública. Metodología: Las cepas provenientes de muestras clínicas fueron analizadas mediante técnicas tradicionales (Kirby-Bauer y epsilometría) y automatizadas, además de técnicas colorimétricas, inmunocromatográficas y moleculares (RPC y PFGE). Resultados: Se detectó la presencia de los genes blaoxa-48 y blaoxa-232 con una resistencia inusual, tanto a carbapenémicos (ertapenem, imipenem y meropenem) como a cefalosporinas (cefepime, cefotaxima y ceftazidima), además de piperacilina/tazobactam y temocilina. Se detectaron dos subtipos por PFGE, siendo predominante el clon CL-Kpn-Spe-329 (93,8%) con dos mecanismos de resistencia identificados: carbapenemasa y β-lactamasa de espectro extendido (BLEE). Conclusión: Ante esta alerta epidemiológica es necesario unificar criterios existentes en la red asistencial nacional para la oportuna detección, vigilancia y control de posibles brotes de cepas productores de oxacilinasa tipo OXA-48.
•A. pleuropneumoniae infection with an isolate of moderate virulence was evaluated.•Specific antibodies, acute phase proteins and inflammatory cytokines were examined.•IL-6, serum A amyloid, ...C-reactive protein and haptoglobin were over-expressed.•Our results are of interest in the study of the pathogenesis of this disease.
Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia (PCP), causes significant economic losses associated mainly with growth stunting of animals. Although serotypes can be distinguished according to their virulence, most of the studies are focused in A. pleuropneumoniae infections with virulent serotypes. There is little information regarding the role of acute phase proteins (APPs) and proinflammatory cytokines in infections with isolates of mild or moderate virulence. Thus, the present study aims to evaluate the kinetics of infection with an A. pleuropneumoniae serotype 6 (Ap6) field isolate of moderate virulence and the changes in the serum concentration of specific antibodies and different APPs and proinflammatory cytokines. Control animals showed no clinical signs or lesions throughout the study. Infected animals showed increased rectal temperature, respiratory distress and depression from 24hpi, and typical gross and microscopic lesions of PCP from 6hpi onwards. Ap6 was isolated from nasal swabs of four out of five inoculated animals at 24hpi, and from nasal swabs, tonsil and lung samples from all inoculated animals at 72hpi. Specific antibodies against Ap6 or changes in the serum concentration of IL-1β, IL-10 and TNF-α were not detected throughout the study. The serum concentration of IL-6 increased from 6hpi as well as serum A amyloid, C-reactive protein and haptoglobin from 24hpi onwards. Our results highlight the onset of the acute phase response after the infection with a field isolate of A. pleuropneumoniae of moderate virulence from 24hpi onwards which may be of interest in the study of the pathogenesis of this disease.
Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new ...therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)
-resorcinarene conjugates derived from LfcinB (20-25): RRWQWR and BF (32-34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)
-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents.
Objective. To describe a standardized flow cytometry protocol for the relative and absolute quantification of hematopoietic cell subpopulations from normal bone marrow, and to evaluate the expression ...of different lineage-specific cell markers with a reactivity associated to cell differentiation to be used as part of the routine quality control in cytometry laboratories. Materials and methods. The immunophenotypical analysis of different cell subpopulations was done with samples from normal bone marrow using a panel of monoclonal and polyclonal antibodies useful in the characterization of acute leukemias with four different fluorescences, by means of a protocol that combines cell labeling of membrane and cytoplasm antigens. Expression analysis was done in terms of mean fluorescence intensity (MFI). Fluorescent beads at a known concentration were added for calculating the absolute count of cells. Results. The antibody panel used allowed the identification and quantification of different normal leukocyte subpopulations of lymphatic and myeloid origin, including CD34+ stem cells and more differentiated cell populations in the granulocytic, monocytic, and erythroid cell lines. We established reference values for cell populations and cell marker expression ranges as part of routine quality control of cytometry laboratories. Conclusion. Immunophenotypic patterns identified as well as absolute and relative reference values for the different normal leukocyte populations from bone marrow can be used by cytometry laboratories as a basis for establishing reference parameters in phenotypic analyses of hematologic neoplasia. Key words: multiparametric flow cytometry, immunophenotype, hematologic neoplasia, normal bone marrow, reference values, quality control.
Objetivo. Evaluar tres tratamientos (lagunaje, fotocatálisis con TiO2 y desinfección química) para la inactivación de coliformes totales y Escherichia coli presentes en agua residual doméstica ...empleada para riego agrícola. Materiales y métodos. El agua residual fue caracterizada por medio de análisis físicos, químicos y microbiológicos. Posteriormente fue sometida a un tratamiento de lagunaje facultativo (TLF), pos tratamiento fotocatalítico (PTFTiO2/UV) y pos tratamiento químico (PTQ NaClO). Valorando la capacidad desinfectante de cada uno de ellos para inactivar coliformes totales y E. coli. A continuación se procesaron tres nuevos lotes de agua residual y se utilizaron para realizar pruebas de riego a escala de laboratorio por 30 días, empleando como modelo plantas de Lactuca sativa variedad Batavia y evaluando la concentración inicial y final de los dos grupos. Resultados. El PTFTiO2/UV fue significativamente superior que TLFLF y el PTQ NaClO (p<0,0001) obteniendo 100% de inactivación para coliformes y E. coli a los 30 minutos de irradiación a escala de reactor. Respecto a las pruebas de riego de L. sativa se demostró que al utilizar el agua tratada por PTFTiO2/UV no se presentó contaminación con E. coli y coliformes a los 30 días de proceso. Por el contrario en las plantas regadas con agua tratada por TLF y PTQ NaClO se observó un incremento en las dos poblaciones generando un problema de contaminación de las hortalizas al finalizar la prueba de laboratorio. Conclusión. La fotocatálisis heterogénea TiO2 fue un método eficaz para la reducción de coliformes y E.coli en aguas residuales domésticas.
Se evaluó un sistema discontinuo secuencial compuesto por células de Bacillus licheniformis y Saccharomyces cerevisiae para producción de etanol, utilizando en la segunda fase del proceso, un ...hidrolizado de almidón de papa, obtenido con el uso de células de B. licheniformis. Ambos microorganismos fueron inmovilizados en matriz de alginato de calcio al 3,2% y 2,5% (p/v), observando que a estas concentraciones se retiene la mayor cantidad de células (26x10(6) y 10x10(7) UFC/g) y permite la difusión de los productos, obteniendo 3,3 g/L de azúcares reductores y 642 UA/L (unidades amilolíticas) para B. licheniformis y 0,866% (v/v) de etanol con S. cerevisiae. Mediante un diseño factorial 22 se seleccionaron las condiciones de operación a escala de reactor para la producción del hidrolizado, encontrando que al cultivar a B. licheniformis con 3 v.v.m. y 150 r.p.m. se produjeron 3,7 g/L de azúcares reductores y 669 UA/L a las 4 horas de proceso. El hidrolizado se caracterizó por cromatografía HPLC determinando que es rico en oligómeros, dextrinas y que tiene baja concentración de glucosa y maltosa. El uso del hidrolizado para la producción de etanol, generó porcentajes bajos (0,47% y 0,74% v/v), tanto en células libres como inmovilizadas, respectivamente.
We evaluated a sequential discontinuous system composed by Bacillus licheniformis and Saccharomyces cerevisiae forethanol production. For the second phase of the process potato starch hydrolyzed were ...used, which was obtained from B.licheniformis cells. Both microorganisms were immobilized in a calcium alginate matrix of 3,2% and 2,5% (w/v), wherewas observed that these concentrations retained the majority of the cells (26x106 and 10x107 UFC/g) and alloweddissemination of its products, gaining 3.3 g/L of reducing sugars and 642 AU/L (units Amylolytic) for B. licheniformis and0,866% (v/v) ethanol with S. cerevisiae. By means of a 22 factorial design were selected operating conditions at a reactorscale for production of hydrolyzed, finding that by cultivating B. licheniformis with 3 v.v.m. and 150 r.p.m. there were 3.7g/L of reducing sugars and 669 AU/L after 4 hours of the process. The hydrolyzed was characterized using HPLCchromatography, which determined that it is rich in oligomers and dextrin, and it has low concentration of glucose andmaltose. The use of hydrolyzed for ethanol production, generated low percentages (0,47% and 0,74% v/v) in free andimmobilized cells respectively.
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